Mixture of Propionibacterium jensenii and Lactobacillus sp. with antimicrobial activities for the use as natural preservation system

ABSTRACT

The invention concerns a mixture of bacteria. Said mixture is a non starter culture which is free from metabolites and comprises at least one first bacterium selected from the species &lt;i&gt; Propionibacterium jensenii &lt;/i&gt; and at least one second bacterium selected from the genus &lt;i&gt; Lactobacillus &lt;/i&gt;. Furthermore, food, feeding stuff and medicaments comprising such a mixture, a method for manufacturing and storing such goods and the use of the mixture to inhibit fungi and bacteria are provided.

CROSS REFERENCES TO RELATES APPLICATIONS

This application claims the priority of the European patent applicationNo. 01 125 464.6, filed Nov. 6, 2001, the disclosure of which isincorporated herein by reference in its entirety.

TECHNICAL FIELD

The invention concerns bacteria suitable for the preservation of goods,in particular a mixture of bacteria.

BACKGROUND ART

Outgrowth of undesired micoorganisms in food products can becounteracted by different ways. Beside physical and chemical treatments,biological treatments such as fermentations by naturally resident and/orartificial (“starter”) microorganisms are widely used. They can changethe food conditions in which growth of undesireable organisms is lessfavourable or completely inhibited as is the case in wine, beer, cheeseor yoghourt (Holzapfel et al. 1995; Vogel, 1996). Another system ofbiologically induced food preservation is the supplementation of“protective cultures” acting as “biopreservatives”. Such cultures arethought

-   a) either to grow and competitively supress undesired organisms-   b) or to grow and produce antimicrobial and antifungal agents (e.g.    bacteriocins, organic acids, diacetyl, unknown metabolites)-   c) or to interact by unknown mechanisms with food spoiling    microorganisms.    Since protective cultures generally do not participate in specific    food modification processes as starter cultures, their application    can also be extended to nonfood materials.

Antimicrobial activities of propionibacteria and lactic acid bacteriamake them appropriate for industrial application as biopreservatives.Bio Profit (Valio Ltd., Helsinki, Finland; Wiesby GmbH & Co., Niebüll,Germany), is a commercially available co-culture of Lactobacillusrhamnosus LC705 (DSM 7061) (former Lactobacillus casei subsp. rhamnosus)and Propionibacterium freuden-reichii subsp. shermanii JS (DSM 7067),that is suggested for a controlling of yeasts and moulds (Soumalainenand Mäyrä-Mäkinen, 1999) Both organisms had been cultured together andwere supposed to be used as a cell containing fermentation brothinhibiting the growth of moulds and yeasts in food. Strain Lactobacillusrhamnosus LC705 was protected in 1993 by a European patent (EP 0 576780) to be used as a single strain or in combination with a bacterium ofthe genus Propionibacterium or another strain of the bacterial speciesLactobacillus casei. A second, German patent (DE 199 17 715) describesprotective cultures consisting of lactic acid bacteria and inhibitingthe growth of toxigenic bacteria at temperatures over 7-8° C. Thecultures are suggested for a preservation of food and animal food(feeding stuff) showing only a short shelf-life below 7-8° C. As soon asthe storage temperature increases, the cultures promise to inhibit thegrowth of spoilage bacteria. In contrast, Microgard™ (Wesman Foods,Inc., Beaverton, Oreg., USA) is a commercially available milk productfermented by a P. freuden-reichii ssp. shermanii strain, followed by apasteurisation process. The product promises effectiveness in inhibitingselected food spoilage organisms as well as pathogenic microorganismsknown to cause foodborne illnesses (Daeschel, 1989; Al-Zoreky et al.,1991). Microgard™ is approved by the US Food and Drug Administration.

DISCLOSURE OF THE INVENTION

Hence, it is a general object of the invention to provide an improvedbacteria based inhibitor for the growth of fungi (yeasts, moulds) and/orbacteria as well as mixtures thereof.

Now, in order to implement this and still further objects of theinvention, which will become more readily apparent as the descriptionproceeds, the invention is manifested by the features that are mentionedin the independent claims.

It was unexpectedly found that bacteria selected from the speciesPropionobacterium jensenii and/or the genus Lactobacillus that arenon-starter cultures and free from metabolites are suitable inhibitorsof undesired microorganisms. In particular a mixture of bacteria (alsoreferred to as inhibiting mixture or protective culture)—which is a nonstarter culture—and which is free from metabolites and that comprises atleast one first bacterium selected from the species Propionibacteriumjensenii and at least one second bacterium selected from the genusLactobacillus, is able to inhibit at least one microorganism selectedfrom fungi and/or bacteria as well as mixtures of fungi and bacteriawhich for example are responsible for the deterioration of food andfeeding stuff.

This mixture of bacteria has the great advantage that—althoughmetabolites that hitherto were thought to be responsible for protectiveactivity are absent—the protection is as good or even better than withhitherto known cultures and undesired side effects due to metabolites(e.g. the metabolites can be toxic or also negatively influence thetaste, colour of food etc.) are markedly reduced or even fullyeliminated.

The mixture of bacteria is incorporated into and/or applied onto thesurface of the good to be protected.

The term “metabolites” comprises all products which origin from themetabolism of the bacteria comprised by the mixture.

In this application, bacteria and fungi which are able to deterioratefood, feeding stuff, medicaments are also called “unwantedmicroorganisms”.

The term “a mixture of bacteria free from metabolites” means that onlythe mixture of bacteria (e.g. separated from metabolites at least bycentrifugation, preferably by centrifugation and at least one washingstep), optionally together with a carrier, is incorporated into and/orapplied onto the surface of the good (food, feeding stuff, medicament)to be protected from deterioration.

Furthermore, in connection with this patent application, the term “to beinhibiting, to inhibit etc.” means for example that the growth or alsothe number or the concentration of unwanted microorganisms, for examplein food and/or onto the surface of food comprising the mixture, is lowerthan in food and/or onto the surface of food which does not comprisesuch a mixture.

The term “a mixture which is a non starter culture” means a mixture thatcomprises bacteria which are not or not essentially adapted to thegood—in particular food, feeding stuff, medicaments—to be protected bythe mixture such that they do not or only minimally grow. Minimal growthmeans a growth of up to at most about tenfold.

A preferred mixture is a mixture of bacteria as described above that isobtainable by the process which comprises the following steps:

Firstly, an inhibiting mixture which comprises at least one first and atleast one second bacterium and which mixture is free from metabolites isincorporated into a medium and/or applied onto the surface of a mediumcontained in a first container such that a minimum concentration asdefined further below of each of said at least one first and at leastone second bacteria in this medium and/or on the surface of this mediumresults.

The medium contained in the first and second container (the secondcontainer is described below) in or on which the inhibition of thecontaminants is tested preferably is food or feeding stuff or amedicament, or much preferably solid agar medium.

The at least one first bacterium is selected from the speciesPropionibacterium jensenii and the at least one second bacterium isselected from the genus Lactobacillus.

As a blank, a second container is prepared comprising the same medium assaid first container but no mixture of bacteria.

Then the same number of contaminants are incorporated into the mediumand/or applied onto the surface of the medium contained in said firstand second containers.

The term “contaminants” is equal to the term “unwanted microorganisms”and comprises fungi and/or bacteria as well as mixtures of fungi andbacteria.

The inhibiting effect of the mixture against the contaminants isdetermined by storing the first and second container at a suitabletemperature during a suitable storage time.

In general, said suitable temperature at which this method is performeddepends on the temperature at which a specific good, in particular food,feeding stuff or medicament normally is stored and/or manufactured.

The temperature at which the first and second container are usuallystored is 5-26° C., preferably said temperature is at least selectedfrom the group of approx. 6° C. and/or approx. 12° C. and/or approx. 25°C.

The term “approx.” means 6±1° C., 12±1° C., 25±1° C.

The storage time at said temperature depends on the time during whichthe good (food, feeding stuff or a medicament) normally is stored.

The storage time usually is 7-28 days, preferably said storage time isselected at least from the group of 7 days and/or 14 days and/or 21 daysand/or 28 days.

During said storage time at a specific temperature, the number or theconcentration of contaminants incorporated into the medium and/orapplied onto the surface of the medium contained in the first and secondcontainer is compared.

Based on this comparison (number/concentration of contaminants in saidfirst container at a specific time versus number/concentration ofcontaminants in said second container at that specific time), a firstand a second graph can be drawn in a coordinate system where e.g. thex-axis represents the storage time and the y-axis represents the numberand/or concentration of contaminants. The first graph shows the numberor concentration of the contaminants incorporated into the medium and/orapplied onto the surface of the medium in the first container and thesecond graph shows the same measure in the medium and/or on the surfaceof the medium contained in the second container.

If only single strains or mixtures of strains of the same species orgenus shall be investigated, the above mentioned method can be usedaccordingly.

The mixture is considered to be inhibiting during the stages of thestorage time, if said second graph is above said first graph.

Preferably, the mixture is considered to be inhibiting if the number ofcontaminants in the medium and/or on the surface of the medium containedin the second container is at least about log 1 higher, preferably atleast about log 2, much preferably at least about log 3, more preferablyat least about log 4, even more preferably at least about log 5, mostpreferably at least about log 6 than the number of contaminants in themedium and/or on the surface of the medium contained in said firstcontainer during at least 3 days, preferably at least 7 days, muchpreferably at least 14 days, more preferably at least 21 days, mostpreferably 25 days.

According to a preferred embodiment of the invention, the at least onefirst bacterium mentioned above is Propionibacterium jensenii SM11.

According to another preferred embodiment of the invention, the at leastone second bacterium mentioned above is selected from the groupconsisting of Lactobacillus paracasei, Lactobacillus rhamnosus,Lactobacillus casei; Lactobacillus plantarum, and mixtures thereof.

According to a further, more preferred embodiment of the invention, theat least one second bacterium is selected from one or more strains ofLactobacillus paracasei subsp. paracasei.

According to an even more preferred embodiment of the invention, the atleast one second bacterium is Lactobacillus paracasei subsp. paracaseiSM20 and/or Lactobacillus paracasei subsp. paracasei SM29 and/orLactobacillus paracasei subsp. paracasei SM63, and mixtures thereof.

The ratio between the at least one first bacterium and the at least onesecond bacterium of the mixture—e.g. the ratio of the concentration ornumber of the at least one first bacterium and the concentration ornumber of the at least one second bacterium—preferably amounts from1:100-100:1, preferably 1:10-10:1.

The mixtures described above provide the advantage that microorganismsselected from the group consisting of fungi, bacteria and mixtures offungi and bacteria which are or can be pathogenic for humans and/oranimals and/or which have or can have a spoilage effect—for example onfood, feeding stuff, medicaments—can be inhibited.

For example in case of food—means for preservation such as chemicalprocesses (e.g. the application of food preservatives, salting) and/orphysical processes (e.g. heat, UV-rays, Gamma-rays, x-rays), packagingusing protective gases (e.g. N₂, CO₂) or vacuum packaging can be reducedor totally omitted without reduction of the storage time/shelf life ofthe food.

As a consequence thereof, especially low processed food or also lowprocessed ingredients which for example are added to high processed foodcan be stored during a longer period without biological deterioration oralso can be better exported to other countries.

If desired, the mixture can also be used in connection with thepreservation processes mentioned above.

Using such preservation processes in combination with the mixture, it isa prerequisite that the mixture still can perform its protectivepurpose.

This means for example that after the application of said preservationprocess, the good to be protected still has to comprise at least thefollowing minimum concentrations of viable cells (cfu=colony formingunit).

The minimum concentration of the at least one first or the at least onesecond bacterium in the food in view of protection amounts at least toabout 1×10⁷ cfu/ml, preferably at least to about 1×10⁸ cfu/ml, even morepreferably at least to about 1×10⁹ cfu/ml or at least to about 1×10⁷cfu/g, preferably at least to about 1×10⁸ cfu/g, even more preferably atleast to about 1×10⁹ cfu/g and/or on the surface of the food at least toabout 1×10⁶ cfu/cm², preferably at least to about 1×10⁷ cfu/cm², muchpreferably at least to about 1×10⁸ cfu/cm², even more preferably atleast to about 1×10⁹ cfu/cm².

If higher activity against contaminants is desired, concentrations of atleast about 1×10¹⁰ cfu/ml or at least about 1×10¹⁰ cfu/g and/or at leastabout 1×10¹⁰ cfu/cm² are possible.

The minimum concentrations mentioned above go out from the experimentalobservation that the higher the concentration or number of theprotective culture is, the more the fungi and/or bacteria as well asmixtures thereof are inhibited.

While some protection is already observed if the first and secondbacteria are present in amounts of 1×10⁷ cfu/ml or cfu/g, the protectionis much better if the at least-one first bacterium and the at least onesecond bacterium combined with each other to the mixture of bacteriaamount at least to about 5×10⁷ cfu/ml or at least to about 5×10⁷ cfu/gand/or at least to about 5×10⁶ cfu/cm² for the at least one firstbacterium and at least to about 1×10⁸ cfu/ml or at least to about 1×10⁸cfu/g and/or at least to about 1×10⁷ cfu/cm² for the at least one secondbacterium. Very satisfying results were achieved with at least about1×10⁸ cfu/ml or at least about 1×10⁸ cfu/g and/or at least about 1×10⁷cm² for the at least one first bacterium and at least about 1×10⁸ cfu/mlor at least about 1×10⁸ cfu/g and/or at least about 1×10⁷ cm² for the atleast one second bacterium. Presently preferred are at least about 5×10⁸cfu/ml or at least about 5×10⁸ cfu/g and/or at least about 5×10⁷ cfu/cm²for the at least one first bacterium and at least to about 5×10⁸ cfu/mlor at least about 5×10⁸ cfu/g and/or at least about 5×10⁷ cfu/cm² forthe at least one second bacterium.

The minimum concentrations and preferred concentrations mentioned aboveapply also to medicaments and feeding stuff.

The determination of the surface concentration can be performed by twomethods:

(i) by measuring the applied amount, e.g. in spraying application, orpreferably

(ii) by measuring the concentration of a specific volume with welldefined application surface and comprising the whole diffusion zone ofthe mixture of bacteria.

The term “at least one first bacterium of 1×10⁷ cfu/ml” means that afirst bacterium and optionally one or more other first bacteria whichare different from each other and which all belong to the genusPropionibacterium jensenii are simultaneously present, whereby all saidfirst bacteria together form a concentration of 1×10⁷ cfu/ml. Thisdefinition also applies to equivalent passages in the application.

The minimum concentrations defined above have to be given in any stageafter the completion of the manufacturing process of the food, feedingstuff or medicament.

In any case, the concentration of the mixture must be higher than or atleast equal to the minimum concentration before the beginning of thestorage.

Preferably, food, feeding stuff or medicaments comprise such aconcentration of the bacteria of the mixture that the concentration ofthe bacteria and/or fungi or mixture of bacteria and fungi is kept belowthe requirements mentioned in the food regulation of the respectivecountry where this invention is used.

Preferably, but not necessarily, these minimum concentrations also applyduring the performance of the manufacturing method.

If the concentration of the bacteria of the mixture in the good and/oronto the surface of the good to be protected falls below the minimumconcentration during the performance of the manufacturing method becauseof for example the application of preservation processes which reducethe viable cells (heat, UV-rays etc.) or as a consequence ofconcentration changes during the manufacturing (e.g. by addition offurther ingredients etc.) cells can be added during the performance ofthe manufacturing. In case of a manufacturing method comprising a longlasting step, it is important that the minimum concentration is alreadypresent during said step such as for example during the ripening ofsausages.

If a reduction of the concentration of viable cells takes place duringthe storage such that the required minimum concentration is not givenany more, additional viable cells have to be added if possible.

In general, the concentration of the mixture of bacteria in food,feeding stuff and medicaments can be higher than the minimumconcentrations mentioned above. But the concentrations have to beselected such that the food, feeding stuff or the medicament is notaffected by undesired deteriorations. This means for example that themixture does not influence the sensory or other quality properties ofthe food (e.g. discoloration etc.).

Besides the mixtures described above, the invention also provides amethod for manufacturing e.g. feeding stuff, medicaments, and preferablyfood, comprising such mixtures.

This manufacturing method allows that unwanted microorganisms are notable to grow.

In the following, this method is described by means of food, but thisdoes not exclude that the following teaching can also be applied to themanufacturing of other goods, e.g. feeding stuff and medicaments.

The method comprises the following steps: Firstly, the mixture is addedduring the manufacturing of the food in an amount such that theconcentration of the at least one first bacterium and the at least onesecond bacterium in the food each amounts at least to the minimumconcentration cfu/ml or cfu/g of the food and/or at least to the minimumconcentration cfu/cm² of the surface of the food.

In general, the mixture has to be added at a stage of the manufacturingmethod or prior to the storage such that it can be evenly distributed inthe good and/or onto the surface of the good to be protected.

The mixture preferably is applied at a stage prior to a significantcontamination with unwanted microorganisms.

In a second step of the manufacturing, e.g. after the addition of themixture, one or more parameters of the manufacturing method have to becontrolled such that the concentration of the mixture decreases or,preferably remains constant. While decrease in concentration can be“healed” as described above, significant growth must be avoided.

Such parameters are for example the temperature, pressure or also theingredients of the food etc.

By the term “constant” it is intended—once the mixture is added to thegood, e.g. food or feeding stuff or medicament—that the concentration ofthe mixture remains constant. Such constant mixture preferably is addedin an amount such that at least the minimum concentration as defined inthis application results. By the term “constant”, is also intended anincrease of number or concentration of the bacteria of the mixture bymeans of growth of the bacteria up to at most tenfold.

In case the manufacturing method comprises one or more fermentationsteps, the mixture—if added prior to the fermentation—must not have anegative influence on the microorganisms (e.g. bacteria, moulds, yeastsor mixtures thereof) responsible for said one or more fermentations(e.g. reduction of the fermentation activity).

If the mixture has such a negative influence on these microorganisms,the mixture is added after said one or more critical fermentation stepsor after the completion of the manufacturing method.

Also in manufacturing methods comprising steps that might be affected bythe mixture of bacteria, said bacteria are added after such one or moresteps.

Furthermore, the invention provides a method for storing goods, inparticular feeding stuff, medicaments and preferably food wherein thegrowth of unwanted microorganisms during storage is inhibited.

This method for storage can be used for the storage of e.g. food,feeding stuff or medicaments which are manufactured according to themanufacturing method of the invention or by known manufacturing methods.

Only during the manufacturing method according to the invention, theparameter or parameters of the manufacturing method are controlled suchthat the concentration of the mixture remains constant.

Using other e.g. common manufacturing methods, there is no or onlypartial such control. Therefore, the mixture should be added in suchmanufacturing method at a stage after which the concentration of themixture remains constant.

The method for storing comprises the step of controlling the storageparameter or storage parameters during the storage such that theconcentration of the at least one first and at least one secondbacterium remains constant.

Possible storage parameters are for example the temperature, storageatmosphere around the good, storage atmosphere within the packaging ofthe good etc.

The term “storage” means the time after completion of the manufacturingof the good, e.g. food, feeding stuff or medicament and therefore saidterm for example can also comprise transportation.

The mixture of bacteria according to the invention preferably iscontained in and/or applied onto the surface of food, feeding stuff ormedicaments.

Such food preferably are dairy products, more preferably sour milkproducts.

Furthermore, meat and meat products are also preferred.

Dairy products are for example raw milk, heat-treated or microfiltratedmilk, butter, curdled milk, sour cream, skim milk, cream, yoghurt,buttermilk, curd, cheese, kefir, kumiss, puddings, sour milk,acidophilus sourmilk, langfil (ropy milk), bifidus sourmilk, junket,sour cream butter.

Meat is for example portioned fresh beef or poultry meat or minced meat.

Meat products preferably are ready to eat sausages, pet food, nitriteand nitrate cured meat (pickled raw meat), cooked salt meat, raw sausageetc.

Products like for example delicatessen salads made of meat, fish,molluscs, crustaceans, vegetable, pasta and mixtures thereof, but alsofish, fish products, molluscs, crustaceans, fruits, fruit products,nuts, nut products, and vegetables such as potatoes, vegetable products,corn, corn products can be protected by the mixture.

Possible combinations of the food mentioned above comprising the mixtureaccording to the invention are also felt to fall under the presentinvention.

Feeding stuff preferably are products derived from nuts, corn, gras.

Medicaments which can be protected by the mixture are natural andorganic remedies.

According to one embodiment of the invention, food, feeding stuff ormedicaments are treated with a powder comprising the mixture whichpowder optionally comprises suitable carrier substances. Suitablecarrier substances are for example saccharides, preferablymonosaccharides, disaccharides or their derivatives.

According to a further preferred embodiment of the invention, food,feeding stuff or medicaments are treated with a liquid medium comprisingthe mixture. Such a liquid medium must be designed such that itguarantees the survival of the mixture. It can be for example an aqueousmedium such as a liquid milk product or a physiological sodium chloridesolution.

According to a preferred embodiment of the invention, the mixture isincorporated into the food, feeding stuff or medicament by mixing,spraying etc., whereby by spraying or another suitable type ofapplication such as dipping, the mixture is applied onto the surface offood, feeding stuff or medicament.

In case of dairy products, the mixture preferably is comprised in aliquid, whereby the liquid is added to the good to be protected. Themixture can also be added as a deep freezed or freeze-dried concentrate.

The mixture according to the invention that is preferably incorporatedinto and/or applied on the surface of food, feeding stuff ormedicaments, more preferably into and/or on those specified above,allows in particular the inhibition of bacteria such as Listeriainnocua, Listeria monocytogenes, Salmonella typhimurium, Staphylococcusaureaus, Escherichia coli, Serratia liquefaciens, Citrobacter freundii,Klebsiella pneumoniae, Enterobacter cloacae, Enterococcus faecalis,Bacillus subtilis, Bacillus cereus, Bacillus anthracis and mixtures ofthese bacteria, lower eukaryotes such as algae and mixtures of lowereukaryotes, fungi such as Candida magnoliae, Candida parapsilosis,Candida silvicola, Candida valida, Candida pulcherrima, Candidareukaufii, Candida krusei, Candida sp., Sporobolomyces salmonicolor,Zygosaccharomyces bailii, Penicillium sp., mixtures of these fungi aswell as mixtures of the bacteria and fungi mentioned above as well asmixtures of the bacteria and fungi and lower eukaryotes mentioned above.

The invention is not considered to be limited to the bacteria, fungi,food, feeding stuff and medicaments explicitly mentioned in thisapplication.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be better understood and objects other than those setforth above will become apparent when consideration is given to thefollowing detailed description thereof. Such description makes referenceto the annexed drawings, wherein:

FIG. 1 shows a composition of 82 isolates on MRS agar originating fromraw milk, cheese, yoghurt, black olives, salami as well as mais and grassilage showing antifungal activities in an agar spot assay and beingidentified with API 50 CHL. ^(a) identified as Lactobacillus paracaseisubsp. paracasei analysing the 16S rRNA gene.

FIG. 2 shows a 2% agarose gel of the amplification of a 290-bp productobtained using the casei-group-specific oligonucleotides in conjunctionwith primer Y2 (Table 2). (A) Lactobacillus paracasei subsp. paracaseiSM20; (B) Lactobacillus paracasei subsp. paracasei SM29; (C)Lactobacillus paracasei subsp. paracasei SM63; (D) Lactobacillus caseiDSM 20011^(T); (E) Lactobacillus paracasei subsp. paracasei DSM 5622^(T)and (F) Lactobacillus rhamnosus DSM 20021^(T); 1, 4, 7, 10, 13, 16,casei/Y2; 2, 5, 8, 11, 14, 17, para/Y2; 3, 6, 9, 12, 15, 18, rham/Y2;19, negative control; kb, linear DNA kb-ladder; pUC,pUC19-DNA/MspI(HpaII) marker.

FIG. 3: Levels of yeasts (Candida pulcherrima 1-50/13) in 5 differentbatches of a food model with different protective cultures, stored at 6°C. (♦) 4.9×10⁸ cfu/g Propionibacterium jensenii SM11 and 1.2×10⁸ cfu/gLactobacillus paracasei ssp. paracasei SM20; (▴) 3.2×10⁸ cfu/gPropionibacterium jensenii SM11 and 1.1×10⁸ cfu/g Lactobacillusparacasei ssp. paracasei SM29; (▪) 4.1×10⁸cfu/g Propionibacteriumjensenii SM11 and 9.1×10⁷ cfu/g Lactobacillus paracasei subsp. paracaseiSM63; (□) 2.7×10⁸ cfu/g Propionibacterium jensenii SM11 and 2.1×10⁸cfu/g Lactobacillus plantarum SM17; (⋄) 2.7×10⁸ cfu/g Propionibacteriumjensenii SM11 and 9.3×10⁷ cfu/g Lactobacillus plantarum SM39; (●) noprotective culture. Number of yeasts are mean values of duplicates.

FIG. 4: Levels of yeasts (Candida pulcherrima 1-50/13, Candida magnoliae1-35/1, Candida parapsilosis 4-5/1 and Zygosaccharomyces bailii 1-48/1)in 7 different batches of yoghurt with different protective culturesstored at 6° C. (⋄) 1.5×10⁷ cfu/g Propionibacterium jensenii SM11 and4.3×10⁷ cfu/g Lactobacillus paracasei ssp. paracasei SM20; (♦) 5.5×10⁷cfu/g Propionibacterium jensenii SM11 and 1.7×10⁸ cfu/g Lactobacillusparacasei ssp. paracasei SM20; (□) 2.0×10⁷ cfu/g Propionibacteriumjensenii SM11 and 3.8×10⁷ cfu/g Lactobacillus paracasei ssp. paracaseiSM29; (▪) 2.8×10⁸ cfu/g Propionibacterium jensenii SM11 and 1.7×10⁸cfu/g Lactobacillus paracasei ssp. paracasei SM29; (Δ) 1.6×10⁷ cfu/gPropionibacterium jensenii SM11 and 1.7×10⁷ cfu/g Lactobacillusparacasei ssp. paracasei SM63; (▴) 8.1×10⁷ cfu/g Propionibacteriumjensenii SM11 and 1.6×10⁸ cfu/g Lactobacillus paracasei ssp. paracaseiSM63; (●) no protective culture. Number of yeasts are mean values ofduplicates.

MODES FOR CARRYING OUT THE INVENTION EXAMPLES

Material and Methods

Bacterial Strains and Media

Bacterial strains and fungi used in this study are listed in Table 1.

TABLE 1 Microbial strains used in this study. Culture conditions StrainRelevant characteristics Temp (° C.) Medium Reference Propionibacteriaand lactobacilli Propionibacterium jensenii SM11 Antimicrobial activity32 NLB LME^(a) Lactobacillus casei DSM 20011^(τ) Type strain 30 MRSDSM^(b) Lactobacillus paracasei subsp. paracasei SM20 Antimicrobialactivity 32 MRS This study SM29 Antimicrobial activity 32 MRS This studySM63 Antimicrobial activity 32 MRS This study DSM 5622^(τ) Type strain30 MRS DSM Lactobacillus plantarum SM17 Antimicrobial activity 32 MRSThis study SM39 Antimicrobial activity 32 MRS This study Lactobacillusrhamnosus DSM 20021^(τ) Type strain 37 MRS DSM Other strains Bacilluscereus DSM 31^(τ) Type strain 30 BHI DSM Bacillus subtilis 168 30 BHILME Enterococcus faecalis DS5 37 BHI LME Staphylococcus aureaus VF4 37BHI LME Listeria innocua L17 37 BHI LME Listeria monocytogenes M1 37 BHILME Citrobacter freundii SG84 30 BHI LME Enterobacter cloacae SG95 30BHI LME Escherichia coli B 37 BHI LME SG63 37 BHI LME Klebsiellapneumoniae SG89 30 BHI LME Salmonella typhimurium ATCC 14028 37 BHIATCC^(c) Serratia liquefaciens SG64 30 BHI LME Candida sp. 1-50/15 25 YMMiescher (1999) Candida krusei 3-69/2 25 YM Miescher (1999) Candidamagnoliae 1-35/1 25 YM Miescher (1999) Candida parapsilosis 4-5/1 25 YMMiescher (1999) Candida pulcherrima 1-50/13 25 YM Miescher (1999)Candida reukaufii 4-73/4 25 YM Miescher (1999) Candida silvicola 4-42/125 YM Miescher (1999) Candida valida 1-48/8 25 YM Miescher (1999)Sporobolomyces salmonicolor 3-46/2 25 YM Miescher (1999)Zygosaccharomyces bailii 1-48/1 25 YM Miescher (1999) Penicillium sp.2-21/7 25 YM Miescher (1999) 2-21/12 25 YM Miescher (1999) 2-52/2 25 YMMiescher (1999) 3-58/3 25 YM Miescher (1999) ^(a)Laboratory for FoodMicrobiology, Zurich, Switzerland ^(b)German Collection ofMicroorganisms and Cell Cultures, Braunschweig, Germany ^(c)AmericanType Culture Collection, Manassas, VA, USA ^(τ)Type strain

Propionibacterium strains were grown in NLB broth that consisted of 1%trypticase soy broth without dextrose (BBL Microbiology Systems,Cockeysville, Md., USA), 1% yeast extract and 1% sodium lactate syrup(Sigma Chemical Co., St. Louis, Mo., USA) as described by Grinstead andBarefoot (1992). Lactobacilli were propagated in MRS broth with 0.1%Tween 80 (Biolife, Italy). All other strains were grown in BHI broth(Biolife, Italy) at growth conditions given in Table 1. Bacterialcultures were maintained as frozen stocks at −80° C. in broth containing30% glycerol. Yeasts and moulds were cultured in YM medium (Difco,Detroit, Mich., USA) and maintained at 4° C. on solid agar medium.

Commercial starters for yoghurt fermentation were obtained from DaniscoCultor Niebüll GmbH, Germany, consisting of different strains ofLactobacillus bulgaricus and Streptococcus thermophilus.

Protective cultures were produced by growing the strains separately insupplemented whey permeate (SWP1) that consisted of 5.5% whey permeate(mmi, Switzerland), 1% yeast extract (Becton Dickinson, Md., USA) and 2%casein hydrolysate (Merck, Germany) for 72 h at 32° C. The cells wereharvested by centrifugation, washed once or twice in 0.85% NaCl andresuspended in tyndallized skim milk (Difco, Detroit, Mich., USA) or in0.85% NaCl in a necessary concentration ranging from 10- to 100-fold.Tyndallized skim milk was previously heat treated three times at 90° C.for 30 min on three following days.

A scale up and a process optimisation was performed by growing the cellsseparately in a 8-1 or a 19-1 bioreactor (type L1523; Bioengineering,Switzerland). at 32° C., with an agitation of 250 rpm using 2% inoculum.Fermentation was performed in supplemented whey permeate (SWP2) thatconsisted of 5.5% whey permeate, 1% yeast extract and 2% glucose. The pHwas controlled at 6.0 adding 3 M NaOH. Simultaneously with the pHcontrol, a feed of nutrients was achieved with 20% yeast extract and 20%glucose. The inoculum was grown in supplemented whey permeate (SWP1) at32° C. for 72 h without agitation. Samples were withdrawn from thefermentations periodically and analysed for cell concentrations andoptical density at 650 nm. Lactobacilli were grown for 72 h,propionibacteria for 90 h. The cells were harvested, washed andconcentrated as described above.

Isolation of Lactic Acid Bacteria

10 g of food sample was stomached for 2 minutes with 90 ml ofdilution-buffer (0.85% NaCl, 0.1% peptone), serially diluted and platedon MRS agar with 0.1% Tween 80. Milk samples were directly diluted andplated. The plates were incubated under anaerobic conditions at 37° C.for 3 days. From each food sample, a variety of different colonies waschosen for a screening for antimicrobial activities.

Screening Methods for Antimicrobial Activities

Two methods were used:

(1) Agar spot assay: The producer culture was spot inoculated onto anagar plate according to Grinstead and Barefoot (1992) and incubated atoptimal growth conditions (Table 1). Each plate was overlaid with 6 mlof tempered soft agar (0.6%) containing 0.2 ml of an indicator culturewhich had grown up to a density of 0.2. The optical density of bacteriawas measured at 650 nm, that of yeasts and moulds at 580 nm. Agarmedium, soft agar medium and incubation of plates was chosen appropriateto the test and indicator organisms. Inhibition of the indicator lawnwas monitored after 24 to 48 h for bacteria, fungi were daily scored forinhibition, after 3 days at 25° C.

(2) Well diffusion test: Wells of a diameter of 7 mm were cut to agarplates and filled with cell-free supernatants of bacterial cultures asdescribed by Daeschel (1992). After complete diffusion of the liquid,soft agar containing an indicator organism was poured over the plates todemonstrate inhibitory activity as described above. Agar medium, softagar medium and incubation of plates was chosen appropriate to the testand indicator organisms.

Characterisation and Identification of Bacterial Isolates

Isolates grown on MRS agar showing antimicrobial activities wereGram-stained and examined microscopically for cellular morphology.Furthermore catalase activity was tested by spotting colonies with 30%hydrogen peroxide and a staining of spores was done. A preliminarystrain identification was done using API 50 CHL (bioMérieux SA, Marcyl'Etoile, France). Additionally, proteolysis was determined in MRS agarwith 5% and 10 % skim milk and production of organic acids was analysedenzymatically (Boehringer Mannheim, Germany; kit used for D/L-lacticacid: E1 112 821/kit used for acetic acid: E0 148 261). Furthermore,growth conditions were determined in MRS broth at different temperaturesfor 72 h and with salinities of 5% and 10% at 32° C. for 72 h.

Identification of selected strains by partial 16S rDNA sequencing

Amplification of 16S rDNA was carried out using directly a colony of thecorresponding strain. PCR was performed with universal oligonucleotidesbak4 (Goldenberger, 1997) and bak11w (Greisen et al., 1994 modified byDasen et al. 1998) using 40 cycles. Annealing was achieved at 56° C. for30sec and extension occurred with Taq DNA polymerase (Amersham PharmaciaBiotech, Uppsala, Sweden) at 72° C. for 2 min, DNA strand separation wasperformed at 95° C. for 5 sec. Amplified DNA was purified using theGFX′M PCR DNA and Gel Band Purification Kit (Amersham Pharmacia Biotech,Uppsala, Sweden). Automated cycle sequencing was done with theTerminator Ready Reaction Mix (Amersham Pharmacia Biotech, Uppsala,Sweden) and oligonucleotides targeting the 16S rDNA (SEQ ID NOS 1-11,respectively, in order of appearance)(Table 2).

TABLE 2 Oligonucleotides targeting the 16S rDNA. Name Sequence CommentReference Y2 5′-TGGTCAQACGAACGCTAGGCCCC-3′ Conserved 16S rDNA^(a) Younget al. (1991) casei 5′-TGCACTGAGATTCGACTTAA-3′ Lactobaciilus casci 16SrDNA Ward and Timmins (1999) para 5′-CACCGAGATTCAACATGG-3′ Lactobacillusparacasei subsp. Ward and Timmins (1999) paracasei 16S rDNA rham5′-TGCATCTTGATTTAATTTTG-3′ Lactobacillus rhamnosus 16S rDNA Ward andTimmins (1999) bak4 5′-AGGAGGTGATCCARCCGCA-3′ Conserved 16S rDNA Greisenet al. (1994) bak11w 5′-AGTTTGATCMTGGCTCAG-3′ Conserved 16S rDNAGoldenberger (1997) eub338 5′-ACTCCTACGGGAGGCAGC-3′ Conserved 16S rDNAAmann et al. (1995) uni515 5′-ACCGCGGCTGCTGGCAC-3′ Conserved 16S rDNALane (1991) uni785 5′-GGMTTAGATACCCTGGTAGTCC-3′ Conserved 16S rDNA Amannet al. (1995) uni1088 5′-CGTTAAGTCCCGCAACCGAGC-3′ Conserved 16S rDNAAmann et al. (1995) uni1392 5′-GTACACACCGCCCGTCA-3′ Conserved 16S rDNALane (1991) ^(a)corresponding to a conserved region of most of the knownbacterial 16S rDNA sequences

35 cycles were performed as follows: 96° C. for 10 sec, 50° C. for 30sec, 60° C. for 4 min. Nucleotide sequencing of both strands from clonedDNA was performed by the dideoxy chain termination method (Sanger etal., 1977) with primer walking using the BigDye Terminator CycleSequencing Ready Reaction Kit and the ABI PRISM ABI 310 Genetic Analyserapparatus (Applied Biosystems, Foster City, Calif., USA) for analysis.DNA sequence analysis, sequence alignments, and sequence databasesearching were conducted with programs contained within the SequenceAnalysis Software Package (version 10.0) licensed from the GeneticsComputer Group (University of Wisconsin, Madison, Wis., USA).

Sequences were compared by the algorithm of Pearson and Lipman (1988)(FastA and TFastA) with sequences in the GenEMBL database copy.

Differentiation of Lactobacillus casei, Lactobacillus racasei andLactobacillus rhamnosus by Polymerase Chain Reaction

Amplification of 16S rDNA was carried out as described by Ward andTimmins (1999) with some modifications. A 290-bp fragment of the 16SrDNA was amplified using species-specific primers (casei, para, rham) inconjunction with primer Y2 corresponding to a conserved region of the16S rDNA (Table 2). Each 50-μl reaction contained 2.5 U of Taqpolymerase, 10 mM Tris-HCl (pH 9.0), 1.5 mM MgCl₂, 50 mM KCl, 1 mM dNTPSet, 1 μM each primer and one colony of the corresponding strain. Taqpolymerase, buffers and dNTP Set for PCR were obtained from AmershamPharmacia Biotech (Uppsala, Sweden). Oligonucleotides were synthesisedby Microsynth (Balgach, Switzerland). The temperature program was asdescribed by Young et al. (1991): 300 s at 93° C., 35 cycles of (45 s at93° C., 45 s at 62° C. and 120 s at 72° C.) and 300 s at 72° C. with afinal cooling to 4° C. Amplification products were separated on a 2%agarose gel.

Investigation of Antimicrobial Activities in Agar Plates

Agar plates were prepared, supplemented with Propionbacterium jenseniiSM11 or an antimicrobial Lactobacillusor a combination of both. Thecultures were previously grown separately for 72 h at 32° C. insupplemented whey permeate (SWP1), centrifugated, washed once andresuspended (1:1) in 0.85% NaCl and then serially diluted indilution-buffer (0.85% NaCl, 0.1% peptone). Portions of 20 ml of agarwere prepared with 10% of each dilution to give a final level of 10⁵,10⁶, 10⁷ and 10⁸ cfu/ml. Agar plates were poured and after solidifying,portions of approximately 2-5 μl of broth cultures of the indicatorstrains were spot inoculated on the plates. YM agar was used for yeastsand moulds and BHI agar for bacteria. Triplex of the agar plates werestored at 6° C., 12° C. and 25° C. for up to 4 weeks and the growth ofindicator organisms was controlled weekly for 6° C. or daily for 12° C.and 25° C. As a control, agar plates without supplementation oflactobacilli and propionibacteria were prepared as well as agar plateswith an addition of 1 g/l Ka-Sorbat (EU-Richtlinie, Nr. 95/2/HG).

Investigation of Antifungal Activities in a Food Model

A food model was set up with portions of 50 g tyndallized skim milk(Difco, Detroit, Mich., USA) that were fermented with a commercialyoghurt culture. Test samples were additionally inoculated with aprotective culture (1% of a 100-fold concentrated culture ofPropionibacterium jensenii SM11 and 1% of a 100-fold concentratedculture of an antifungal Lactobacillus) to give a level of 10⁸ cells/gof each. The cultures were previously washed twice and resuspended intyndallized skim milk. To get comparable results, the control sample wasmixed with 2% tyndallized skim milk. The fermentation was done for about5 hours at 42° C. until the pH reached 4.6. All samples werecontaminated with Candida pulcherrima 1-50/13 at a final level of 10²cfu/g. Additionally a control sample was prepared without a yeastscontamination. Samples were stored at 6° C. for 7 weeks and protectivepropionibacteria and lactobacilli as well as yeasts were enumeratedweekly. Simultaneously, pH was controlled weekly.

Inhibition of Fungi in Yoghurt by Protective Cultures

Portions of 200 g milk enriched with 5$ cream (35% fat, 2.5% proteins),2% skim milk powder and 5% sugar were heat treated at 90° C. for 10 minand then fermented with a commercial yoghurt culture. Test samples wereadditionally inoculated with a protective culture (0.1% or 1% of a100-fold concentrated culture of Propionibacterium jensenii SM11 and0.1% or 1% of a 100-fold concentrated culture of an antifungalLactobacillus-strain) to give a level of 10⁷ or 10⁸ cfu/g of each.Protective cultures were previously washed twice and resuspended intyndallized skim milk. The fermentation was done for about 5 hours at42° C. until the pH reached 4.6. The yoghurt was contaminated with a mixof Candida pulcherrima 1-50/13, Candida magnoliae 1-35/1, Candidaparapsilosis 4-5/1 and Zygosaccharomyces bailii 1-48/1 at a final levelof 10² cfu/g. Additionally a control sample was prepared without ayeasts contamination. The yoghurt was stored at 6° C. for 4 weeks andyeasts were enumerated weekly. Simultaneously, pH was controlled weekly.At day 1 and after 3 weeks of storage, protective propionibacteria andlactobacilli were enumerated and furthermore after 3 weeks lactate andacetate were determined enzymatically (Boehringer Mannheim, Germany; kitused for D/L-lactic acid: E1 112 821/kit used for acetic acid: E0 148261).

Scale Up of the Application of a Protective Culture in Yoghurt

Scale up trials with yoghurt comprising the protective culture were alsoperformed as described above.

Inhibition of Yeasts on a Cheese Surface by Protective Cultures

Equal cubes (2.5×4.8×8 cm) of 100 g hard cheese (Gruyère) were prepared.To avoid a contamination, with microorganisms of the cheese surface, 3-5mm of the cheese's rind were previously cut off. One batch of cheese wassoaked with each cube's side in a protective culture (5 ml of a 20-foldconcentrated culture of Propionibacterium jensenii SM11 and 5ml of a20-fold concentrated culture of an antifungal Lactobacillus-strain) togive a level of 10⁸ cfu/g cheese surface of each. Protective cultureswere previously washed twice and resuspended in 0.85% NaCl. Two otherbatches of cheese were prepared with 10-fold and 100-fold dilutedprotective cultures. Onto the surface of the cubes of all three batches,about 3 ml of the respective concentrated protective culture was thusapplied. Then, the cheeses were contaminated with 1 ml (10⁴ cfu/ml) of amix of Candida pulcherrima 1-50/13, Candida magnoliae 1-35/1, Candidaparapsilosis 4-5/1 and Zygosaccharomyces bailii 1-48/1 to reach a finallevel of 10² cfu/g cheese surface. The surface of the cheeses wasdefined as a layer of about 3 mm from the outside of the cube. A controlsample was prepared without an addition of yeasts. The cheeses weresealed in sterile plastic bags and stored for 3 weeks at 6° C. At day 1and after 3 weeks of storage, the level of protective propionibacteriaand lactobacilli was determined as well as the number of yeasts.

Inhibition of Listeria by a Protective Culture in a Food Model

Two food models were set up to test antibacterial activities of aprotective culture against Listeria.

(1) Inhibition of Listeria innocua L17 and Listeria monocytogenes M1 bya Protective Culture in Cream:

200 g of pasteurised half-cream (25% fat, 2.5% proteins) were inoculatedwith Listeria innocua L17 or Listeria monocytogenes M1, respectively, ata final level of 10³ cfu/g and were then divided into two portions of100 g in sterile flasks. One sample was inoculated with a protectiveculture (5% of a 20-fold concentrated culture of Propionibacteriumjensenii SM11 and 5% of a 20-fold concentrated culture of Lactobacillusparacasei subsp. paracasei SM20) to give a level of 10⁸ cfu/g of each.Protective cultures were previously-washed twice and resuspended intyndallized skim milk. To get comparable concentrations, 10% oftyndallized skim milk were added to the control sample. All samples werestored at 6° C. for 4 weeks. The pH and growth of Listeria wascontrolled weekly, after 1 day, and after 4 weeks the level ofprotective culture was checked as well. A second batch was prepared asdescribed and stored at 25° C. for 3 weeks. Similarly, the numbers ofprotective cultures and of Listeria were determined at the beginning andat the end of storage.

(2) Inhibition of Listeria innocua L17 by protective cultures in mincedmeat: Portions of 100 g of minced meat were mixed with a protectiveculture (5% of a 20-fold concentrated culture of Propionibacteriumjensenii SM11 and 5% of a 20-fold concentrated culture of anantimicrobial Lactobacillus-strain) to give a level of 10⁸ cfu/g ofeach. Protective cultures were previously washed twice and resuspendedin 0.85% NaCl. The meat was contaminated with Listeria innocua L17 at afinal concentration of 10³ cfu/g and was then stored at 6° C. for 8weeks. Additionally a control sample was prepared without protectiveculture. To get comparable concentrations, 10% of 0.85% NaCl were addedto this sample. Protective propionibacteria and lactobacilli as well asListeria innocua L17 were enumerated weekly, pH was controlled at theend of the storage period.

Results

Antifungal Activities in Lactobacillus sp.

A preliminary screening for antimicrobial activities was performed with1424 isolates on MRS agar originating from raw milk, cheese, yoghurt,black olives, sour dough, salami, as well as maize and gras silage. Atotal of 82 strains showed activities in an agar spot assay against thefungi listed in Table 1. These isolates were further characterised byGram-staining, catalase-activity, micoscropy and forming of spores. Asdepicted in FIG. 1 the species belonging to the Lactobacilluscasei-group showed predominantly antifungal activities with a share of24%. A preliminary identification with API 50 CHL classified theseisolates as Lactobacillus casei, Lactobacillus paracasei subsp.paracasei and Lactobacillus rhamnosus. Furthermore the speciesLactobacillus plantarum showed high inhibitory activities towards thetested fungi. A total of 6% of the antifungal isolates appeared to becocci and 18% remained unidentified using fermentation patterns.

Identification and Characterisation of Three High Antifungallactobacilli

Three isolates with high antifungal activities were further identifiedusing sequencing analysis of the 16S rDNA. Studying carbohydratemetabolisms, they were previously identified as Lactobacillus paracaseisubsp. paracasei (SM20 and SM29) and Lactobacillus curvatus (SM63).Sequencing analysis of the 16S rDNA of these strains revealed thespecies Lactobacillus paracaseisubsp. paracasei for strain SM20 with anidentity of 100% in 1519 nucleotides overlapping with the partialsequence of the 16S rDNA of Lactobacillus paracasei JCM8130 (D79212) andwith the partial 16S rDNA-sequence of a Lactobacillus casei (D86517). Atotal of 1520 nucleotides were identified from the 16S rDNA of strainSM20. 1528 nucleotides sequenced from strain SM29 showed an identity of99.9% in 1520 nucleotides overlapping with the partial sequence of the16S rRNA gene of a Lactobacillus casei (D86517) and with the partial 16SrDNA-sequence of Lactobacillus paracasei JCM8130 (D79212). Surprisingly,1515 nucleotides identified of the 16S rRNA gene of strain SM63 showedalso an identity of 99.9% in 1493 nucleotides overlapping with the 16SrRNA gene of a Lactobacillus casei (D86517) and with the partialsequence of the 16S rDNA of Lactobacillus paracasei JCM8130 (D79212).

Since members of the casei-group form a very homologous group concerningphenotypic criteria as well as sequence data of the 16S rRNA gene, Wardand Timmins (1999) developed a simple PCR approach to differentiate thespecies Lactobacillus casei, Lactobacillus paracasei and Lactobacillusrhamnosus based on the reclassification by Collins et al. (1989). FIG. 2shows a specific product of 290 bp for strains SM20, SM29 and SM63amplified in a polymerase chain reaction with the Lactobacillusparacasei-specific primer used in conjunction with primer Y2, bothtargeting the 16S rRNA gene. The type strains of Lactobacillus casei(DSM 20011^(T)) and Lactobacillus paracasei subsp. paracasei (DSM5622^(T)) also revealed a 290-bp band with the corresponding primers.Surprisingly, Lactobacillus rhamnosus (DSM 20021^(T)) did not show thespecific band after polymerase chain reaction with Y2 and rham.Furthermore, no products were observed if this primer pair (Y2/rham) wasused with the strains to be identified. Thus, the isolates SM20, SM29and SM63 were identified as Lactobacillus paracasei subsp. paracasei.Table 3 shows a summary of the identification of the three isolates aswell as their characterisation.

TABLE 3 Identification and characterisation of strains SM20, SM29, SM63and SM11. Fermentation Strain Origin Micoscropy Spores Acid producedfrom^(a) products^(b) Growth conditions SM20 Raw short rods negativeRibose, Galactose, D-, L-Lactate 15–45° C. milk in chains, D-Glucose,D-Fructose, (Opt.: 32–37° C. Gram- D-Mannose, Mannitol, positive^(e)Sorbitol, N Acetyl glucosamine, Amygdaline, Arbutine, Escuilne,Salicine, Cellobiose, Maltose, Lactose, Saccharose, Trehalose, Inuline,Melezitose, β Gentiobiose, D-Turanose, D-Tagatose SM29 Salami short rodsnegative Ribose, Adonitol, Galactose, D-, L-Lactate 15–45° C. in chains,D-Glucose, (Opt: 32–37° C. Gram- D-Fructose, D-Mannose, positiveRhamnose, Dulcitol, Mannitol, Sorbitol, α Metyhl-D-mannoside, αMethyl-D-glucoside, N Acetyl glucosamine, Amygdaline, Arbutine,Esculine, Salicine, Cellobiose, Maltose, Lactose, Saccharose, Trehalose,Inuline, Melezitose, β Gentiobiose, D-Turanose, D-Tagatose SM63 Rawshort rods negative Ribose, Galactose, D-, L-Lactate 15–45° C. milk inchains, D-Glucose, D-Fructose. (Opt.: 32–37° C. Gram- D-Mannose,Mannitol, positive Sorbitol, N Acetyl glucosamine, Maltose, Lactose,Trehalose, Melezitose, D- Tagatose SM11 Raw pleomorphic Erythritol,D-Arabinose, Acetate, 15–37° C. milk rod- Ribose, Adonitol, Galactose,Propionate^(f) (Opt.: 30–32° C. shaped, D-Glucose, Gram- D-Fructose,D-Mannose, positive Lactose, D-Arabitol. NaCl Proteolytic CatalaseIdentification Strain Tolerance activity activity API 50 CHLSequencing^(c) PCR^(d) SM20 10%^(g) negative negative LactobacillusLactobacillus Lactobacillus paracasei subsp. paracasei paracaseiparacasei subsp. paracasei ^(l) subsp. and Lactobacillus paracasei caseiSM29  5%^(h) negative negative Lactobacillus LactobacillusLacotobacillus paracasei subsp. paracasei paracasei paracaseisubsp. paracasei subsp. and Lactobacillus paracasei casei SM63  5%^(i)negative negative Lactobacillus Lactobacillus Lactobacillus curvatusparacasei paracasei subsp. paracasei subsp. and Lactobacillus paracaseicasei SM11  5%^(k) negative positive Propionibacterium iensenii^(a)determined using API 50 CHL ^(b)determined enzymatically (BoehringerMannheim, Germany; kit used for D/L-lactic acid: E1 112 821/kit used foracetic acid: E0 148 261) ^(c)sequencing analysis of the 16S rDNA ^(d)PCRapproach modified according to Ward and Timmins (1999) ^(e)long rods inchains in bioreactor cultures ^(f)Miescher (1999) ^(g)well growth at asalinity of 5% and fairly well growth at 10% ^(h)well growth at asalinity of 5% NaCl and very weak growth at 10% ^(i)well growth at asalinity of 5% and weak growth at 10% ^(k)fairly well growth at asalinity of 5% NaCl and very weak growth at 10% ^(l)underlined speciesindicates the final identification of the corresponding strain

Collins et al. (1989) described another subspecies of Lactobacillusparacasei, namely Lactobacillus paracasei subsp. tolerans. Regarding theacid production observed by Collins et al. (1989), the isolates SM20,SM29 and SM63 belong to the subspecies paracasei.

Secretion of Inhibitory Substances into Broth Medium

Lactobacillus paracasei subsp. paracasei SM20, Lactobacillus paracaseisubsp. paracasei SM29, Lactobacillus paracasei subsp. paracasei SM63,Lactobacillus plantarum SM17 and Lactobacillus plantarum SM39 wereexamined for their ability to secret inhibitory substances into brothmedium. Therefore strains were either grown in MRS broth or in SWP1medium. No antagonistic activity was detected in the cell-freesupernatants using a well diffusion test. Even a concentration step byultrafiltration (molecular weight cut-off of 3'000 Da) did not revealany detectable activity (data not shown).

Antifungal Activities in Agar Plates

A total of 18 lactobacilli were tested for their antifungal activitiesalone or in combination with the high-antifungal Propionibacteriumjensenii SM11. Therefore, the strains were incorporated, aftercentrifugation and a washing step in concentrations from 10⁵ to 10⁸cfu/ml in YM agar plates on which the fungi listed in Table 1 werespotted in corresponding levels from 10¹ to 10⁷ cfu/ml. The plates werestored in different batches at 6° C., 12° C. and 25° C. The growth ofyeasts was controlled weekly or daily, respectively. Although, thelactobacilli as well as the Propionibacterium strain showed only weakinhibitory activities using them alone (data not shown) theircombination revealed high antagonistic values at 6° C. The highestactivities were detected with the following lactobacilli in combinationwith Propionibacterium jensenii SM11: Lactobacillus paracasei subsp.paracasei SM20, Lactobacillus paracasei subsp. paracasei SM29 andLactobacillus paracasei subsp. paracasei SM63. Weak activities wereobserved with Lactobacillus plantarum SM17 and Lactobacillus plantarumSM39 in conjunction with strain SM11. A cell number of 10⁸ cfu/ml of theLactobacillus-strain and 10⁸ cfu/ml of the Propionibacterium-strain wasnecessary for high inhibitory activities. Table 4 indicates fiveexamples of the antifungal activities observed at 6° C. with aconcentration of 10⁸ cfu/ml (10⁷ cfu/ml for Lactobacillus plantarumSM17) against spot-inoculated yeasts in a density of 10⁶ cfu/ml andagainst spot-inoculated 3-day cultures of moulds.

TABLE 4 Antifungal activities of selected protective culturesincorporated in agar plates after a storage of 21 days at 6° C., or 7days at 12° C. Protective cultures^(b) SM20/SM11 SM29/SM11 SM63/SM11SMI7/SM11 SM39/SM11 control Ka-Sorbat Indicatororganisms^(a) (pH3.83)^(c) (pH 3.98) (pH 3.93) (pH 4.53) (pH 4.59) (pH 5.88) (lg/l)^(d)Storage at 6° C./21 d Candida magnoliae 1-35/1 + +/− + ++ ++ ++ ++Candida parapsilosis 4-5/1 − − − − − (+/−) − Zygosacharomyces bailil1-48/1 − − − − − − − Candida silvicola 4-42/1 (+/−) (+/−) (+/−) ++ ++ ++++ Candida valida 1-48/8 (+/−) (+/−) (+/−) ++ ++ + − Candida pulcherrima1-50/13 (+/−) − (+/−) ++ ++ ++ ++ Candida reukaufii 4-73/4 − − − ++ ++++ ++ Candida sp. 1-50/15 (+/−) (+/−) (+/−) ++ ++ ++ ++ Sporobolomycessalmonicolor 3-46/2 − − − − − ++ − Candida krusei 3-69/2 − − − (+/−)(+/−) (+/−) − Penicillium sp. 2-21/7 − − − n.d. n.d. ++ n.d. Penicilliumsp. 2-21/12 − − − n.d. n.d. ++ n.d. Penicillium sp. 2-52/2 − − − n.d.n.d. ++ n.d. Penicillium sp. 3-58/3 − − − n.d. n.d. ++ n.d. Storage at12° C./7 d Candida magnoliae 1-35/1 (+/−) + + + + ++ n.d Candidaparapsilosis 4-5/1 − (+/−) − +/− +/− ++ n.d Zygosacharomyces bailil1-48/1 − (+/−) (+/−) (+/−) − +/− n.d Candida silvicola 4-42/1 (+/−)(+/−) (+/−) (+/−) (+/−) ++ n.d Candida valida 1-48/8 − + + ++ + ++ n.dCandida pulcherrima 1-50/13 − ++ + ++ ++ ++ n.d Candida reukaufii 4-73/4− + − ++ ++ ++ n.d Candida sp. 1-50/15 − ++ ++ ++ ++ ++ n.dSporobolomyces salmonicolor 3-46/2 − − − + +/− +/+ n.d Candida krusei3-69/2 + ++ ++ ++ ++ ++ n.d SM20: Lactobacillus paracasei subsp.paracasei SM20 (10⁸ cfu/ml) SM29: Lactobacillus paracasei subsp.paracasei SM29 (10⁸ cfu/ml) SM63: Lactobacillus paracasei subsp.paracasei SM63 (10⁸ cfu/ml) SM17: Lactobacillus plantarum SM17 (10⁷cfu/ml) SM39: Lactobacillus plantarum SM39 (10⁷ cfu/ml) SM11:Propionibacterium jensenii SM11 (10⁸ cfu/ml) n.d.: not determined^(a)spot-inoculation with 10⁶ cfu/ml ^(b)incorporated in agar plates^(c)pH measured on agar plates after a storage of 21 days at 6° C.^(d)concentration according to EU-Richlinie, Nr. 95/2/EG + normal colony(≈25% inhibition) ++ strong colony (≈0% inhibition) +/− weak colony(≈50% inhibition) (+/−) very weak colony (≈75% inhibition) − no colony(≈100% inhibition)

Interestingly, using Ka-Sorbat, a weaker inhibitory activity wasobserved at 6° C. than with the antimicrobial cultures of this study(strains SM20, SM29 or SM63 in combination with strain SM11). Table 4further depicts, that after a storage of 7 days at 12° C. a clearinhibitory activity was observed for Lactobacillus paracasei subsp.paracasei strains SM20, SM29 and SM63 and a weak for Lactobacillusplantarum strains SM17 and SM39 in conjunction with Propionibacteriumjensenii SM11. At 25° C. only weak inhibitory activities were observedthat disappeared after a few days (data not shown). Testing theantifungal activities of 2 or 3 lactobacilli (Lactobacillus paracaseisubsp. paracasei strains SM20, SM29 and SM63) in conjunction withPropionibacterium jensenii SM11, an increase in inhibition was observed(Table 5) in comparison with the trials in which 1 Lactobacillus strainin combination with SM11 was used (Table 4). A total inhibition of theyeasts was reached using Lactobacillus paracasei subsp. paracaseistrains SM20, SM29 or SM63 in conjunction with Propionibacteriumjensenii SM11 and 1 g/l Ka-Sorbat (Table 5). The antifungal activity ofthe bacterial strains was increased with the food preservative.

TABLE 5 Antifungal activities of selected protective culturesincorporated in agar plates after a storage of 21 days at 6° C.Protective cultures^(b) SM20/ SM20/SM29/ SM20/SM63/ SM29/SM63/SM29/SM63/ SM20/SM11/ SM29/SM11/ SM63/SM11/ SM11 SM11 SM11 SM11Ka-Sorbat^(d) Ka-Sorbat Ka-Sorbat control Indicatororganisms^(a) (pH4.27)^(c) (pH 4.32) (pH 4.33) (pH 4.10) (pH 4.67) (pH 4.80) (pH 4.75)(pH 6.04) Candida (+/−) (+/−) +/− (+/−) − − − ++ magnoliae 1-35/1Candida − − − − − − − − parapsilosis 4-5/1 Zygosacharomyces − − − − − −− − bailii 1-48/1 Candida − − − − − − − ++ silvicola 4-42/1 Candida − −(+/−) − − − − (+/−) valida 1-48/8 Candida − − (+/−) − − − − ++pulcherrima 1-50/13 Candida − − − − − − − ++ reukaufii 4-73/4 Candida −− (+/−) − − − − + sp. 1-50/15 Sporobolomyces − − − − − − − +/−salmonicolor 3-46/2 Candida − − − − − − − − krusei 3-69/2 SM20:Lactobacillus paracasei subsp. paracasei SM20 (10⁸ cfu/ml) SM29:Lactobacillus paracasei subsp. paracasei SM29 (10⁸ cfu/ml) SM63:Lactobacillus paracasei subsp. paracasei SM63 (10⁸ cfu/ml) SM11:Propionibacterium jensenii SM11 (10⁸ cfu/ml) n.d.: not determined^(a)spot-inoculation with 10⁶ cfu/ml ^(b)incorporated in agar plates^(c)pH measured on agar plates after a storage of 21 days at 6° C. ^(d)1g/l, concentration according to EU-Richtlinie, Nr. 95/2/EG ++ strongcolony (≈0% inhibition) + normal colony (≈25% inhibition) +/− weakcololny (≈50% inhibition) (+/−) very weak colony (≈75% inhibition) − nocolony (≈100% inhibition)

Antibacterial Activities of Protective Cultures in Agar Plates

Lactobacillus paracasei subsp. paracasei strains SM20, SM29 and SM63were further tested for their ability to inhibit Gram-positive andGram-negative bacteria alone or in combination with Propionibacteriumjensenii SM11. Therefore, the strains were incorporated aftercentrifigation and a washing step in a concentration of 10⁸ cfu/ml inBHI agar plates on which overnight cultures of the correspondingbacteria were spotted. One batch of the plates was stored at 6° C. andthe growth of bacteria was controlled weekly, a second was incubated at25° C. and was daily scored for inhibition. Again, the lactobacilli aswell as the Propionibacterium strain showed weak inhibitory activitiesusing them alone (data not shown) but their combination revealed highantagonistic values (Table 6).

TABLE 6 Antibacterial activities of selected protective culturesincorporated in agar plates after a storage of 21 days at 6° C. or at25° C. Protective cultures^(b) control SM20/SM11 SM29/SM11 SM63/SM11 (pHIndicatororganisms^(a) (pH 5.45^(c)) (pH 5.48) (pH 5.40) 6.70) Storageat 6° C./21 d Listeria innocua L17 − − − ++ Staphylococcus aureus VF4 −− − (+/−) Escherichia coli B − − − (+/−) Escherichia coli SG63 − − − +/−Sereratia liquefaciens SG64 − (+/−) + ++ Citrobacter freundii SG84 (+/−)(+/−) (+/−) ++ Klebsiella pneumoniae SG89 (+/−) (+/−) +/− ++Enterobacter cloacae SG95 (+/−) (+/−) (+/−) + Enterococcus faecalis DS5− − − + Bacillus subtilis 168 − − − − Bacillus cereus DSM 31^(τ) − − − −Salmonella typhimurium ATCC 14058 − − − ++ Listeria monocytogenes M1 − −− ++ Storage at 25° C./21 d Listeria innocua L17 − − − ++ Staphylococcusaureus VF4 + +/− +/− ++ Escherichia coli B + (+/−) (+/−) ++ Escherichiacoli SG63 +/− − − ++ Sereratia liquefaciens SG64 +/− + +/− ++Citrobacter freundii SG84 (+/−) +/− (+/−) ++ Klebsiella pneumoniae SG89(+/−) +/− + ++ Enterobacter cloacae SG95 (+/−) + + ++ Enterococcusfaecalis DS5 (+/−) (+/−) (+/−) ++ Bacillus subtilis 168 − − − ++Bacillus cereus DSM 31^(τ) − (+/−) (+/−) ++ Salmonella typhimurium ATCC14028 − − − ++ Listeria monocytogenes M1 − − − ++ SM20: Lactobacillusparacasei subsp. paracasei SM20 (10⁸ cfu/ml) SM29: Lactobacillusparacasei subsp. paracasei SM29 (10⁸ cfu/ml) SM63: Lactobacillusparacasei subsp. paracasei SM63 (10⁸ cfu/ml) SM11: Propionibacteriumjensenii SM11 (10⁸ cfu/ml) ^(a)spot-inoculation with an overnightculture ^(b)incorporated in agar plates ^(c)pH measured on agar platesafter a storage of 21 days at 6° C. ++ strong colony (≈0% inhibition)(+/−) very weak colony (≈75% inhibition) + normal colony (≈25%inhibition) − no colony (≈100% inhibition) +/− weak cololny (≈50%inhibition)

Antifungal Activities of Stored Protective Cultures

In order to determine the ability to store the protective cultures, theywere held separately at a 100-fold concentration (resuspended intydallized skim milk) at −80° C. for 15 weeks. Then, the strains wereincorporated in a concentration of 10⁸ cfu/ml in YM agar plates on whichthe yeasts listed in Table 1 were spotted in levels from 10⁴ to 10⁷cfu/ml. The plates were stored at 6° C. and growth of yeasts wascontrolled weekly. As a control, an agar plate was prepared with anaddition of an appropriate amount of skim milk. Table 7 shows in asummary, that the protective cultures consisting of Propionibacteriumjensenii SM11 and a Lactobacillus paracasei subsp. paracasei strain(SM20, SM29 or SM63) still showed unchanged antifungal activities aftera storage of 15 weeks at −80° C.

TABLE 7 Antifungal activities of selected protective cultures after astorage of 15 weeks at −80° C., incorporated in agar plates. Protectivecultures^(b) Indicatororganisms^(a) SM20/SM11 SM29/SM11 SM63/SM11control Candida magnoliae 1-35/1 +/− +/− +/− ++ Candida parapsilosis4-5/1 − − − (+/−) Zygosacharomyces bailii 1-48/1 − − − − Candidasilvicola 4-42/1 − − − ++ Candida valida 1-48/8 + + + + Candidapulcherrima 1-50/13 +/− +/− + ++ Candida reukaufii 4-73/4 − − − ++Candida sp. 1-50/15 + + + ++ Sporobolomyces salmonicolor 3-46/2 − − −+/− Candida krusei 3-69/2 − − − − SM20: Lactobacillus paracasei subsp.paracasei SM20 (10⁸ cfu/ml) SM29: Lactobacillus paracasei subsp.paracasei SM29 (10⁸ cfu/ml) SM63: Lactobacillus paracasei subsp.paracasei SM63 (10⁸ cfu/ml) SM11: Propionibacterium jensenii SM11 (10⁸cfu/ml) n.d.: not determined ^(a)spot-inoculation with 10⁶ cfu/ml^(b)incorporated in agar plates ++ strong colony (≈0% inhibition) +normal colony (≈25% inhibition) +/− weak cololny (≈50% inhibition) (+/−)very weak colony (≈75% inhibition) − no colony (≈100% inhibition)

Antifungal Activities of Protective Cultures in a Food Model

For a preliminary trial, a food model was set up with differentprotective cultures. The antagonistic strains were added to tyndallizedskim milk at initial levels of 10⁸ cfu/g. They were Lactobacillusparacasei subsp. paracasei SM20, Lactobacillus paracasei subsp.paracasei SM29, Lactobacillus paracasei subsp. paracasei SM63,Lactobacillus plantarum SM17 and Lactobacillus plantarum SM39 incombination with Propionibacterium jensenii SM11. Additionally thesamples were inoculated with Candida pulcherrima 1-50/13 at levels of10² cfu/g and then stored at 6° C. As indicated in FIG. 3, the levels ofyeasts increased constantly to 10⁷-10⁸ cfu/g in the samples with theLactobacillus plantarum strains SM17 and SM39 as well as in the controlsample showing the highest values. Samples containing Lactobacillusparacasei subsp. paracasei (SM20, SM29 or SM63) showed significantdifferences. In these samples, Candida pulcherrima 1-50/13 increased upto a level of 10⁴ cfu/g and then remained almost stable for seven weeks.After four weeks samples with Lactobacillus paracasei subsp. paracaseiSM63 showed an increase in yeasts. The level of propionibacteria andlactobacilli neither increased nor decreased during storage. The pH ofsamples with protective cultures did not show any significantdifferences to the control sample (data not shown).

Antifungal Activities in Yoghurt

A further trial was set up with yoghurt and protective culturesconsisting of Lactobacillus paracasei subsp. paracasei strains SM20,SM29 or SM63 in combination with Propionibacterium jensenii SM11.Therefore, a milk enriched with nutrients was used that should favourthe growth of contaminating yeasts. This milk was fermented with acommercial yoghurt culture in the presence of a protective culture. Theprotective cultures were used at concentrations of 10⁷ cfu/g or 10⁸cfu/g, respectively. After fermentation and cooling down, the sampleswere inoculated with a mix of Candida pulcherrima 1-50/13, Candidamagnoliae 1-35/1, Candida parapsilosis 4-5/1 and Zygosaccharomycesbailii 1-48/1 at a level of 10² cfu/g and then stored at 6° C. FIG. 4shows that the levels of yeasts in samples with protective cultures at aconcentration of 10⁷ cfu/g as well as in the control sample increasedconstantly. Nevertheless, the control sample always showed the highestvalues of contaminating yeasts. In all samples containing the protectivecultures at a level of 10⁸ cfu/g no increase of yeasts was observed. Thelevel of yeasts remained stable over a period of 4 weeks. The samephenomenon was observed with concentrations of 1.7×10⁸ cfu/glactobacilli and 5.5×10⁷ cfu/g propionibacteria (Table 11). In allsamples, the levels of protective culture neither increased nordecreased during storage. The pH of samples with protective cultures didnot show any significant differences to the control sample (data notshown). As shown in Table 8, in samples with protective cultures 60-70mg/100 g acetate were enzymatically determined whereas only 4.43 mg/100g were detected in the control sample without protective culture.

TABLE 8 Enzymatically determined concentrations of organic acidsmetabolised in yoghurt with protective cultures. Organic acids AcetateD-Lactate L-Lactate Total lactate Protective cultures (mg/100 g) (g/100g) (g/100 g) (g/100 g) SM20/SM11^(a) 68.84 0.01 0.61 0.62 SM29/SM11^(b)60.95 0.01 0.62 0.63 SM63/SM11^(c) 61.44 0.01 0.65 0.66 control^(d) 4.430.01 0.74 0.75 SM20: Lactobacillus paracasei subsp. paracasei SM20 SM29:Lactobacillus paracasei subsp. paracasei SM29 SM63: Lactobacillusparacasei subsp. paracasei SM63 SM11: Propionibacterium jensenii SM11^(a)SM20, 1.7 × 10⁸ cfu/g; SM11, 5.5 × 10⁷ cfu/g ^(b)SM29, 1.7 × 10⁸cfu/g; SM11, 2.8 × 10⁸ cfu/g ^(c)SM63, 1.6 × 10⁸ cfu/g; SM11, 8.1 × 10⁷cfu/g ^(d)no protective culture

In contrast, the protective culture did not influence the content ofD-lactate, L-lactate as well as total lactate (Table 8).

Using different sensory test, the yoghurts with protective cultures andthe control samples without an additional culture did not show anyperceptible differences after storage periods of 5 and 15 days.

Scale Up of the Application of a Protective Culture to Yoghurt

The scale up trials with yoghurt gave similar results as were obtainedby the trials on laboratory scale.

Antifungal Activities on the Cheese Surface

Table 9 shows the inhibition of a mix of yeasts on cheese surfaces,treated with three different protective cultures at differentconcentrations. The cheeses were all contaminated with yeasts to reach afinal level on the surface of 10² cfu/g. The surface of the cheeses wasdefined as a layer of about 3 mm from the outside of the cube.

TABLE 9 Inhibition of yeasts on cheese surfaces held at 6° C. cfu/g ofyeasts^(a) Time (days) A B C K1 1 5.0 × 10² 3.2 × 10² 4.1 × 10² 21 1.5 ×10² <10² <10² K2 1 5.5 × 10² 1.0 × 10² 4.1 × 10² 21 3.5 × 10² <10² <10²K3 1 2.2 × 10³ 2.7 × 10² 1.3 × 10³ 21 1.3 × 10⁴ <10² 1.0 × 10² K4 1 6.3× 10² 21 8.0 × 10³ A: inoculated with yeasts and protective cultureSM20/SM11 B: inoculated with yeasts and protective culture SM29/SM11 C:inoculated with yeasts and protective culture SM63/SM11 K1:concentration of protective culture: 10⁷–10⁸ cfu/g surface K2:concentration of protective culture: 10⁶–10⁷ cfu/g surface K3:concentration of protective culture: approx. 10⁶ cfu/g surface K4:control, inoculated with yeasts, no protective culture added ^(a)a mixof Candida pulcherrima 1-50/13, Candida magnoliae 1-35/1, Candidaparapsilosis 4-5/1 and Zygosaccharomyces bailii 1-48/1

At concentrations of 10⁸ and 10⁷ cfu/g surface, the protective cultureswere able to inhibit the outgrowth of yeasts totally during a storage of21 days at 6° C. The number of yeasts even declined (K1, K2). A level of3.0×10⁶ cfu/g surface of lactobacilli and 3.0×10⁶ cfu/g surface ofpropionibacteria seemed to be the limit to reach a minimal inhibition ofthe yeasts. Cheese sample K3A showed lower levels of protective cultureand did thus not inhibit the outgrowth of the contaminating yeasts. Thelevels of protective cultures in the surfaces of the cheeses were in thesame range as the cell numbers of the solutions in which the cheeses hadpreviously been soaked (data not shown).

Inhibition of Listeria in Food Models with Protective Cultures

Table 10 summarises the inhibition of Listeria with protective culturesin minced meat and cream.

TABLE 10 Inhibition of Listeria by protective cultures in food modelsheld at 6° C. and 25° C. Food systems Time (days) A B C D cfu/g ofListeria innocua L17 Minced 1 3.3 × 10³ 2.8 × 10³ 2.6 × 10³ 2.7 × 10³meet 28 6.0 × 10² 3.7 × 10² 2.7 × 10² 4.5 × 10² (6° C.) 42 1.7 × 10³<10² 1.5 × 10² <10² 56 5.1 × 10³ 8.2 × 10² <10² 1.5 × 10² Minced 0 4.1 ×10³ 4.9 × 10³ 4.0 × 10³ 3.7 × 10³ meet 2 4.5 × 10⁴ 5.5 × 10³ 3.0 × 10³7.5 × 10³ (25° C.) 5 6.8 × 10⁴ 5.9 × 10³ 3.2 × 10³ 5.5 × 10³ Cream 1 2.7× 10³ 3.2 × 10³ n.d. n.d. (6° C.) 14 1.3 × 10³ 1.2 × 10³ n.d. n.d. 211.1 × 10³ 1.5 × 10² n.d. n.d. 28 1.0 × 10³ <10² n.d. n.d. Cream 1 2.9 ×10² 2.6 × 10² 2.2 × 10² 3.0 × 10² (25° C.) 21 10⁷–10⁸ <10² <10² <10²cfu/g of Listeria monocytogenes M1 Cream 1 1.6 × 10³ 1.7 × 10³ n.d. n.d.(6° C.) 7 2.9 × 10⁷ 1.9 × 10³ n.d. n.d. 14 3.0 × 10⁸ 7.5 × 10² n.d. n.d.A: control, inoculated with Listeria no protective culture added B:inoculated with Listeria and protective culture SM20/SM11 C: inoculatedwith Listeria and protective culture SM29/SM11 D: inoculated withListeria and protective culture SM63/SM11 n.d.: not determined

The two food models were selected to represent high moisture-food withdifferent fat and protein contents and an optimal pH for Listeria.Although Listeria innocua L17 showed good growth after 21 days on BHIagar stored at 6° C. (Table 6), it was not detected in the food models.The viable numbers of the indicator organism, however, decreased in thepresence of a protective culture. Furthermore, these samples stillshowed a nice red meat colour after a storage of 56 days and the controlsample was green to brown and clearly spoiled. The pH of the controlsample was 6.20 after 56 days that one of the samples with protectivecultures in a range of 5.50 to 5.81. The level of propionibacteria andlactobacilli remained unchanged during the storage at 6° C. for a periodof 56 days. A second charge of minced meat with and without protectivecultures was stored at 25° C. After a storage of 5 days, Listeriainnocua L17 was held in check in the samples with protective cultures(3.2 to 5.9×10³ cfu/g) whereas the viable number of the indicatororganism increased to 6.8×10⁴ cfu/g in the control sample. Additionally,the control sample had totally gone bad. The cream stored at 6° C.showed the same phenomenon after 28 days. In the control sample, thenumber of Listeria innocua L17 was still in the same range as in thebeginning (10³ cfu/g) but in the sample with protective culture, theviable number of the indicator organism went down to under 10² cfu/g.The level of lactobacilli and propionibacteria neither increased nordescresed during storage. The pH values in the samples with or withoutprotective culture were in the same range. After a storage of 28 days at6° C. the pH of the cream has gone down to 4.78 in the sample withoutprotective culture and to 4.60 in the sample with protective culture. Incream samples stored at 25° C. Listeria innocua L17 decreased below 10²cfu/g in the presence of protective cultures but went up to a cellnumber of 10⁷-10⁸ cfu/g in the control sample. Similarly the pH of thesesamples changed clearly. The samples with protective cultures were in arange of 3.56-3.68, whereas the control sample still had a pH of 5.80.After 21 days of storage at 25° C., the cell numbers of lactobacilli andpropionibacteria were still in the same range as at the beginning. Thelevels of protective cultures neither increased nor decreased. In thesamples with Listeria monocytogenes M1 as indicator organism a cleardifference in the viable number of the pathogen was observed in thepresence or absence of the protective culture. After a storage of 14days at 6° C., the sample without protective culture contained 3.0×10⁸cfu/g of Listeria monocytogenes M1 whereas in the sample with theprotective culture the number of the indicator organism had evendecreased to 7.5×10² cfu/g (Table 10).

Necessary Concentration of Protective Culture in Food for a SufficientAntimicrobial Activity

To reach a sufficient antimicrobial activity, a certain level oflactobacilli and propionibacteria was necessary. Tables 11 and 12 showthe different trials of this study and the corresponding levels ofprotective culture.

TABLE 11 Levels of protective cultures used in the different trials todetect antifungal activities (cfu/g). Trials Agarplates^(a) Food ModelYoghurt^(b) Yoghurt^(c) Cultures LB PAB LB PAB LB PAB LB PAB SM20/SM1110⁸ 10⁸ 1.2 × 10⁸ 4.9 × 10⁸ 1.7 × 10⁸ 5.5 × 10⁷ 4.3 × 10⁷ 1.5 × 10⁷SM29/SM11 10⁸ 10⁸ 1.1 × 10⁸ 3.2 × 10⁸ 1.7 × 10⁸ 2.8 × 10⁸ 3.8 × 10⁷ 2.0× 10⁷ SM63/SM11 10⁸ 10⁸ 9.1 × 10⁷ 4.1 × 10⁸ 1.6 × 10⁸ 8.1 × 10⁷ 1.7 ×10⁷ 1.6 × 10⁷ SM20: Lactobacillus paracasei subsp. paracasei SM20 SM29:Lactobacillus paracasei subsp. paracasei SM29 SM63: Lactobacillusparacasei subsp. paracasei SM63 SM11: Propionibacterium jensenii SM11LB: lactobacilli PAB: propionibacteria n.d. not determined ^(a)level ofthe protective culture was only determined empirically ^(b)(1% of eachstrain in a 100-fold concentration) ^(c)(0.1% of each strain in a100-fold concentration)

TABLE 12 Levels of protective cultures used in the different trials todetect antibacterial activities (cfu/g). Trials Agarplates^(a) Mincedmeat^(b) Minced meat^(c) Creme^(d) Creme^(e) Cultures LB PAB LB PAB LBPAB LB PAB LB PAB SM20/SM11 10⁸ 10⁸ 1.1 × 10⁹ 6.3 × 10⁹ 4.8 × 10⁸ 2.9 ×10⁸ 9.3 × 10⁸ 3.8 × 10⁹ 1.0 × 10⁹ 5.7 × 10⁹ SM29/SM11 10⁸ 10⁸ 1.2 × 10⁹8.1 × 10⁹ 4.9 × 10⁸ 4.0 × 10⁸ n.d. n.d. n.d. n.d. SM63/SM11 10⁸ 10⁸ 1.6× 10⁸ 8.7 × 10⁹ 1.2 × 10⁸ 3.0 × 10⁸ n.d. n.d. n.d. n.d. SM20:Lactobacillus paracasei subsp. paracasei SM20 SM29: Lactobacillusparacasei subsp. paracasei SM29 SM63: Lactobacillus paracasei subsp.paracasei SM63 SM11: Propionibacterium jensenii SM11 LB: lactobacilliPAB: propionibacteria n.d. not determined ^(a)level of the culture wasonly determined empirically ^(b)minced meat stored at 6° C. withListeria innocua L17 ^(c)minced meat stored at 25° C. with Listeriainnocua L17 ^(d)creme stored at 6° C. with Listeria innocua L17^(e)creme stored at 6° C. with Listeria monocytogenes M1

To reach thus an optimal antifungal activity of the protective culture alevel of at least 1.7×10⁸ cfu/g or cfu/ml of lactobacilli in conjunctionwith 5.5×10⁷ cfu/g or cfu/ml of propionibacteria were necessary (Table11). On the cheese surface, a level of 1.0×10⁷ cfu/g surface oflactobacilli and 5.0×10⁶ cfu/g surface of propionibacteria seemed to bethe minimal concentration to reach a constant satisfactory antifungalactivity (data not shown). For a sufficient antibacterial activity, thelowest level tested was 1.2×10⁶ cfu/g or cfu/ml of lactobacilli inconjunction with 3.0×10⁸ cfu/g or cfu/ml of propionibacteria showingstill good inhibitory properties (Table 12). According to hithertoinvestigations the antimicrobial activities are in the same range as theantifungal.

While there are shown and described presently preferred embodiments ofthe invention, it is to be distinctly understood that the invention isnot limited thereto but may be otherwise variously embodied andpracticed within the scope of the following claims.

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Zusatzstoffverordnung über die in Lebensmitteln zulässigen Zusatzstoffe,vom 26. Juni 1995, Stand am 22. Februar 2000. EidgenössischesDepartement des Innern: Dreifuss. EDMZ, Bern.

DEPOSITED STRAINS

The following strains have been deposited on 18 Sep. 2001 with theDSMZ-Deutsche Sammlung von Mikroorganisment and Zellkulturen GmbH(DSMZ), currently located at Inhoffenstraβe 7 B, 38124 Braunschweig,Germany, under the terms of the Budapest Treaty.

-   -   Strain Propionibacterium jensenii SM11 has been deposited under        the number DSM 14513    -   Strain Lactobacillus paracasei subsp. paracasei SM20 has been        deposited under the number DSM 14514.    -   Strain Lactobacillus paracasei subsp. paracasei SM29 has been        deposited under the number DSM 14515.    -   Strain Lactobacillus paracasei subsp. paracasei SM63 has been        deposited under the number DSM 15416.

PCT Original (for SUBMISSION) - printed on 28.10.2002 01:20:47 PM 0-1Form - PCT/RO/134 (EASY) Indications Relating to DepositedMicroorganism(s) or Other Biological Material (PCT Rule 13bis) 0-1-1Prepared using PCT-EASY Version 2.92 (updated 01.10.2002) 0-2International Application No. 0-3 Applicant's or agent's file reference05365PC 1 The indications made below relate to the depositedmicroorganism(s) or other biological material referred to in thedescription on: 1-1 page 57 1-2 line  7 1-3 Identification of Deposit1-3-1 Name of depositary institution DSMZ-Deutsche Sammlung vonMikroorganismen und Zellkulturen GmbH 1-3-2 Address of depositaryinstitution Mascheroder Weg 1b, D-38124 Braunschweig, Germany 1-3-3 Dateof deposit 20 Sep. 2001 (20.09.2001) 1-3-4 Accession Number DSMZ 145131-4 Additional Indications NONE 1-5 Designated States for Which alldesignated States Indications are Made 1-6 Separate Furnishing ofIndications NONE These indications will be submitted to theInternational Bureau later 2 The indications made below relate to thedeposited microorganism(s) or other biological material referred to inthe description on: 2-1 page 57 2-2 line 10 2-3 Identification ofDeposit 2-3-1 Name of depositary institution DSMZ-Deutsche Sammlung vonMikroorganismen und Zellkulturen GmbH 2-3-2 Address of depositaryinstitution Mascheroder Weg 1b, D-38124 Braunschweig, Germany 2-3-3 Dateof deposit 20 Sep. 2001 (20.09.2001) 2-3-4 Accession Number DSMZ 145142-4 Additional Indications NONE 2-5 Designated States for Which alldesignated States Indications are Made 2-6 Separate Furnishing ofIndications NONE These indications will be submitted to theInternational Bureau later 3 The indications made below relate to thedeposited microorganism(s) or other biological material referred to inthe description on: 3-1 page 57 3-2 line 13 3-3 Identification ofDeposit 3-3-1 Name of depositary institution DSMZ-Deutsche Sammlung vonMikroorganismen und Zellkulturen GmbH 3-3-2 Address of depositaryinstitution Mascheroder Weg 1b, D-38124 Braunschweig, Germany 3-3-3 Dateof deposit 20 Sep. 2001 (20.09.2001) 3-3-4 Accession Number DSMZ 145153-4 Additional Indications NONE 3-5 Designated States for Which alldesignated States Indications are Made 3-6 Separate Furnishing ofIndications NONE These indications will be submitted to theInternational Bureau later 4 The indications made below relate to thedeposited microorganism(s) or other biological material referred to inthe description on: 4-1 page 57 4-2 line 16 4-3 Identification ofDeposit 4-3-1 Name of depositary institution DSMZ-Deutsche Sammlung vonMikroorganismen und Zellkulturen GmbH 4-3-2 Address of depositaryinstitution Mascheroder Weg 1b, D-38124 Braunschweig, Germany 4-3-3 Dateof deposit 20 Sep. 2001 (20.09.2001) 4-3-4 Accession Number DSMZ 154164-4 Additional Indications NONE 4-5 Designated States for Which alldesignated States Indications are Made 4-6 Separate Furnishing ofIndications NONE These indications will be submitted to theInternational Bureau later FOR RECEIVING OFFICE USE ONLY 0-4 This formwas received with the International application: (yes or no) 0-4-1Authorized officer FOR INTERNATIONAL BUREAU USE ONLY 0-5 This form wasreceived by the International Bureau on: 0-5-1 Authorized officer

1. A composition comprising isolated bacteria selected from the groupconsisting of Propionibacterium jensenii SM11 (DSM 14513), Lactobacillusparacasei subsp. paracasei SM20 (DSM 14514), Lactobacillus paracaseisubsp. paracasei SM29 (DSM 14515) and Lactobacillus paracasei subsp.paracasei SM63 (DSM 14516), or mixtures thereof.